1. Field of the Invention
The present invention relates to novel enzyme of trehalase and also to a method of manufacturing sugars by the enzyme. More particularly, it relates to a novel trehalase derived from an alga which belongs to Lobosphaera, Chlorellaceae and also to a method of manufacturing trehalose, 2,2'-dideoxy-.alpha.,.alpha.'-trehalose (hereinafter referred to as 2,2'-dideoxytrehalose) and 2-deoxy-.alpha.,.alpha.'-trehalose (hereinafter referred to as 2-deoxytrehalose) by the enzyme.
2. Description of the Related Art
Trehalose is a kind of nonreducing disaccharide in which two molecules of D-glucose are bonded with the .alpha.,.alpha.'-1,1' linkage. In insects, it is available in their blood and lymph and serves as a stored blood sugar for a mobile energy source. Also, it is said that trehalose exhibits an effect as an antifreezer and hence, insects control in some seasons, its concentration for achieving a resistance against coldness.
Trehalase is an enzyme which hydrolyzes trehalose into two glucose molecules. This enzyme may be found in higher animals (small intestine of rats, pigs, etc.), insects (silkworm, etc.), microorganisms and the like. Various microorganisms are known to produce trehalase, including bacteria such as the Mycobacterium genus (Methods in Enzymology, (1972) 28, 996) the Streptomyces genus (J. Bacteriol. (1968) 96, 105); yeasts such as the Saccharomyces genus (J. Biol. Chem., (1964) 239, 1671); and filamentous fungi such as Aspergillus genus (J. Bacteriol., (1966) 91, 1883), the Neurospora genus (J. Bacteriol., (1973) 115, 582), Humicola (Biochim. Biophys. Acta, (1978) 525, 162) or Phycomyces (Biochim. Biophys. Acta, (1975), 391, 154); and the like. All of those contain trehalase either in cells or in spores. Besides the above, the Trichoderma genus (Can. J. Microbiol., (1978) 24, 1280) and the Chaetomium genus (Examined Japanese Patent Publication 22917/85) have been reported as microorganisms producing trehalase outside the cells. However, there has been no report of trehalase derived from Chlorophyceae (green algae).
With regard to the use of trehalose, applications as a sweetening agent causing little dental caries, as an agent acting against dental caries, as a promoter of the promulgation of Bifidus bacteria, and as a novel material for food have been proposed (Japanese Laid Open Publication 63/240758). In addition, there is a report that, when a certain amount of trehalose is added to beverages or food materials containing water, denaturation of protein in beverages and food material can be prevented (National Publication of International Application 02/503864). Further, in the field of pharmaceuticals, it has been reported that trehalose may be added as a protective agent for protein to inhibit a decrease in enzymatic activity thereby permitting a storage of the enzyme-labelled antibody for long periods (Japanese Laid Open Publication 60/149972). Moreover, it has also been reported that trehalose is useful for improving the stability of cells upon storage for stabilizing vaccines and for making trehalose derivatives useful as anticancer agents (Japanese Laid Open Publication 62/174094). For example, it is known that 2-deoxyglucose inhibits the chain formation of glycoproteins and glycolipids as a metabolic antagonist for glucose and, whereby growth of fibroblast is inhibited and anticancer action results.
In manufacturing trehalose, it is known that trehalose may be produced in vitro by means of an enzymatic reaction, or by a method in which trehalose is accumulated in and outside the cells of microorganisms. With regard to the production of trehalose from microorganisms, a method of preparing same from baker's yeast containing large quantities of trehalose is known. For example, a method in which a compressed yeast is extracted with 95% (v/v) ethanol to give trehalose (J. Am. Chem. Soc., (1950) 2059) and one in which baker's yeast is extracted with 90% (v/v) ethanol to give trehalose (Nippon Nogei Kagaku Kaishi, vol. 27, (1953) 412) are known. With regard to an enzymatic method, a method in which maltose is treated with maltose phosphorylase and trehalose phosphorylase to manufacture trehalose has been reported (Examined Japanese Publication 60998/88).
With regard to methods for manufacturing 2,2'-dideoxytrehalose and 2-deoxytrehalose, a method by chemical synthesis (Nouveau Journal De Chimie, (1980) 4, 59) and a method by utilizing metabolism of enzymes (Biochimica et Biophysica Acta, (1984) 803, 284 and Biochimica et Biophysica Acta, (1969) 184, 77) have been described.
However, in the above-mentioned trehalase which is known or is commercially available, there are disadvantages that units produced by the microorganism are low and that the efficiency in the condensation reaction (reverse reaction) from glucose to trehalose is extremely low.
Especially in a method of preparing trehalose by extraction of baker's yeast, a large amount of an organic solvent is necessary and, in addition, complicated steps are necessary for removing the contaminants. In a method of manufacturing trehalose by treating maltose with maltose phosphorylase and trehalose phosphorylase which is a well-known method utilizing enzymes, there are so many disadvantages that the manufacturing steps are complicated and that the manufacture of the enzymes--maltose phosphorylase and trehalose phosphorylase--requires high cost and many difficulties.
There is another method in which trehalose excreted from cells is purified (Japanese Laid Open Publication 05/211882). However, in any of those methods, the manufacturing cost is high and, accordingly, the use is limited.
In a method of manufacturing 2,2'-dideoxytrehalose and 2-deoxytrehalose by means of chemical synthesis, there are disadvantages that the steps are complicated and the yield is low, while in a method by means of metabolism of enzymes, there are disadvantages that the production is low and the purifying steps are complicated.